Cellular carcinogenesis in preleukemic conditions:drivers and defenses.

Acute myeloid leukemia (AML) arises from preleukemic conditions. We have investigated the pathogenesis of typical preleukemia, myeloproliferative neoplasms, and clonal hematopoiesis. Hematopoietic stem cells in both preleukemic conditions harbor recurrent driver mutations; additional mutation provokes further malignant transformation, leading to AML onset. Although genetic alterations are defined as the main cause of malignant transformation, non-genetic factors are also involved in disease progression. In this review, we focus on a non-histone chromatin protein, high mobility group AT-hook2 (HMGA2), and a physiological p53 inhibitor, murine double minute X (MDMX). HMGA2 is mainly overexpressed by dysregulation of microRNAs or mutations in polycomb components, and provokes expansion of preleukemic clones through stem cell signature disruption. MDMX is overexpressed by altered splicing balance in myeloid malignancies. MDMX induces leukemic transformation from preleukemia via suppression of p53 and p53-independent activation of WNT/ß-catenin signaling. We also discuss how these non-genetic factors can be targeted for leukemia prevention therapy.

Preleukemic single-cell landscapes reveal mutation-specific mechanisms and gene programs predictive of AML patient outcomes.

Acute myeloid leukemia (AML) and myeloid neoplasms develop through acquisition of somatic mutations that confer mutation-specific fitness advantages to hematopoietic stem and progenitor cells. However, our understanding of mutational effects remains limited to the resolution attainable within immunophenotypically and clinically accessible bulk cell populations. To decipher heterogeneous cellular fitness to preleukemic mutational perturbations, we performed single-cell RNA sequencing of eight different mouse models with driver mutations of myeloid malignancies, generating 269,048 single-cell profiles. Our analysis infers mutation-driven perturbations in cell abundance, cellular lineage fate, cellular metabolism, and gene expression at the continuous resolution, pinpointing cell populations with transcriptional alterations associated with differentiation bias. We further develop an 11-gene scoring system (Stem11) on the basis of preleukemic transcriptional signatures that predicts AML patient outcomes. Our results demonstrate that a single-cell-resolution deep characterization of preleukemic biology has the potential to enhance our understanding of AML heterogeneity and inform more effective risk stratification strategies.

Molecular and clinical aspects relevant for counseling individuals with clonal hematopoiesis of indeterminate potential.

Clonal hematopoiesis of indeterminate potential (CHIP) has fascinated the medical community for some time. Discovered about a decade ago, this phenomenon links age-related alterations in hematopoiesis not only to the later development of hematological malignancies but also to an increased risk of early-onset cardiovascular disease and some other disorders. CHIP is detected in the blood and is characterized by clonally expanded somatic mutations in cancer-associated genes, predisposing to the development of hematologic neoplasms such as MDS and AML. CHIP-associated mutations often involve DNA damage repair genes and are frequently observed following prior cytotoxic cancer therapy. Genetic predisposition seems to be a contributing factor. It came as a surprise that CHIP significantly elevates the risk of myocardial infarction and stroke, and also contributes to heart failure and pulmonary hypertension. Meanwhile, evidence of mutant clonal macrophages in vessel walls and organ parenchyma helps to explain the pathophysiology. Besides aging, there are some risk factors promoting the appearance of CHIP, such as smoking, chronic inflammation, chronic sleep deprivation, and high birth weight. This article describes fundamental aspects of CHIP and explains its association with hematologic malignancies, cardiovascular disorders, and other medical conditions, while also exploring potential progress in the clinical management of affected individuals. While it is important to diagnose conditions that can lead to adverse, but potentially preventable, effects, it is equally important not to stress patients by confronting them with disconcerting findings that cannot be remedied. Individuals with diagnosed or suspected CHIP should receive counseling in a specialized outpatient clinic, where professionals from relevant medical specialties may help them to avoid the development of CHIP-related health problems. Unfortunately, useful treatments and clinical guidelines for managing CHIP are still largely lacking. However, there are some promising approaches regarding the management of cardiovascular disease risk. In the future, strategies aimed at restoration of gene function or inhibition of inflammatory mediators may become an option.

[Clonal hematopoiesis].

Clonal hematopoiesis (CH) harboring a leukemia-related mutation has been recently found in about 10% of healthy elderly individuals, which has been attracting attention. Although most people with CH do not develop hematological malignancies, some may develop hematological malignancies 10 times more frequently than age-matched controls. On the other hand, compared to age-matched controls, the probability of developing cardiovascular diseases in people with CH is 2-fold, which is thought to shorten the life expectancy. Moreover, one out of four patients with solid cancer and one out of two patients with aplastic anemia, whose mutation profiles overlap with but are distinct from common mutations identified with CH of elderly people, harbor CH. The study of CH has just begun, and there are many unknowns. In an aging society of unprecedented proportions, which is also attracting attention from the society, the establishment of a new research field that investigates CH in the near future is likely.

Mitochondrial calcium homeostasis in hematopoietic stem cell: Molecular regulation of quiescence, function, and differentiation.

Hematopoiesis is based on the existence of hematopoietic stem cells (HSC) with the capacity to self-proliferate and self-renew or to differentiate into specialized cells. The hematopoietic niche is the essential microenvironment where stem cells reside and integrate various stimuli to determine their fate. Recent studies have identified niche containing high level of calcium (Ca2+) suggesting that HSCs are sensitive to Ca2+. This is a highly versatile and ubiquitous second messenger that regulates a wide variety of cellular functions. Advanced methods for measuring its concentrations, genetic experiments, cell fate tracing data, single-cell imaging, and transcriptomics studies provide information into its specific roles to integrate signaling into an array of mechanisms that determine HSC identity, lineage potential, maintenance, and self-renewal. Accumulating and contrasting evidence, are revealing Ca2+ as a previously unacknowledged feature of HSC, involved in functional maintenance, by regulating multiple actors including transcription and epigenetic factors, Ca2+-dependent kinases and mitochondrial physiology. Mitochondria are significant participants in HSC functions and their responsiveness to cellular demands is controlled to a significant extent via Ca2+ signals. Recent reports indicate that mitochondrial Ca2+ uptake also controls HSC fate. These observations reveal a physiological feature of hematopoietic stem cells that can be harnessed to improve HSC-related disease. In this review, we discuss the current knowledge Ca2+ in hematopoietic stem cell focusing on its potential involvement in proliferation, self-renewal and maintenance of HSC and discuss future research directions.

The Bone Marrow Niche in B-Cell Acute Lymphoblastic Leukemia: The Role of Microenvironment from Pre-Leukemia to Overt Leukemia.

Genetic lesions predisposing to pediatric B-cell acute lymphoblastic leukemia (B-ALL) arise in utero, generating a clinically silent pre-leukemic phase. We here reviewed the role of the surrounding bone marrow (BM) microenvironment in the persistence and transformation of pre-leukemic clones into fully leukemic cells. In this context, inflammation has been highlighted as a crucial microenvironmental stimulus able to promote genetic instability, leading to the disease manifestation. Moreover, we focused on the cross-talk between the bulk of leukemic cells with the surrounding microenvironment, which creates a "corrupted" BM malignant niche, unfavorable for healthy hematopoietic precursors. In detail, several cell subsets, including stromal, endothelial cells, osteoblasts and immune cells, composing the peculiar leukemic niche, can actively interact with B-ALL blasts. Through deregulated molecular pathways they are able to influence leukemia development, survival, chemoresistance, migratory and invasive properties. The concept that the pre-leukemic and leukemic cell survival and evolution are strictly dependent both on genetic lesions and on the external signals coming from the microenvironment paves the way to a new idea of dual targeting therapeutic strategy.

iPSC modeling of stage-specific leukemogenesis reveals BAALC as a key oncogene in severe congenital neutropenia.

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can evolve to acute myeloid leukemia (AML). Mutations in CSF3R and RUNX1 are frequently observed in CN patients, although how they drive the transition from CN to AML (CN/AML) is unclear. Here we establish a model of stepwise leukemogenesis in CN/AML using CRISPR-Cas9 gene editing of CN patient-derived iPSCs. We identified BAALC upregulation and resultant phosphorylation of MK2a as a key leukemogenic event. BAALC deletion or treatment with CMPD1, a selective inhibitor of MK2a phosphorylation, blocked proliferation and induced differentiation of primary CN/AML blasts and CN/AML iPSC-derived hematopoietic stem and progenitor cells (HSPCs) without affecting healthy donor or CN iPSC-derived HSPCs. Beyond detailing a useful method for future investigation of stepwise leukemogenesis, this study suggests that targeting BAALC and/or MK2a phosphorylation may prevent leukemogenic transformation or eliminate AML blasts in CN/AML and RUNX1 mutant BAALC(hi) de novo AML.

MDMX acts as a pervasive preleukemic-to-acute myeloid leukemia transition mechanism.

MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). We report that MDMX overexpression increases preleukemic stem cell (pre-LSC) number and competitive advantage. Utilizing five newly generated murine models, we found that MDMX overexpression triggers progression of multiple chronic/asymptomatic preleukemic conditions to overt AML. Transcriptomic and proteomic studies revealed that MDMX overexpression exerts this function, unexpectedly, through activation of Wnt/ß-Catenin signaling in pre-LSCs. Mechanistically, MDMX binds CK1α and leads to accumulation of ß-Catenin in a p53-independent manner. Wnt/ß-Catenin inhibitors reverse MDMX-induced pre-LSC properties, and synergize with MDMX-p53 inhibitors. Wnt/ß-Catenin signaling correlates with MDMX expression in patients with preleukemic myelodysplastic syndromes and is associated with increased risk of progression to AML. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML transition mechanism in different genetically driven disease subtypes, and reveals Wnt/ß-Catenin as a non-canonical MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

Human pluripotent stem cells identify molecular targets of trisomy 12 in chronic lymphocytic leukemia patients.

Identifying precise targets of individual cancers remains challenging. Chronic lymphocytic leukemia (CLL) represents the most common adult hematologic malignancy, and trisomy 12 (tri12) represents a quarter of CLL patients. We report that tri12 human pluripotent stem cells (hPSCs) allow for the identification of gene networks and targets specific to tri12, which are controlled by comparative normal PSCs. Identified targets are upregulated in tri12 leukemic cells from a cohort of 159 patients with monoclonal B cell lymphocytosis and CLL. tri12 signaling patterns significantly influence progression-free survival. Actionable targets are identified using high-content drug testing and functionally validated in an additional 44 CLL patient samples. Using xenograft models, interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor is potent and selective against human tri12 CLL versus healthy patient-derived xenografts. Our study uses hPSCs to uncover targets from genetic aberrations and apply them to cancer. These findings provide immediate translational potential as biomarkers and targets for therapeutic intervention.

Cooperating, congenital neutropenia-associated Csf3r and Runx1 mutations activate pro-inflammatory signaling and inhibit myeloid differentiation of mouse HSPCs.

Patients with the pre-leukemia bone marrow failure syndrome called severe congenital neutropenia (CN) have an approximately 15% risk of developing acute myeloid leukemia (AML; called here CN/AML). Most CN/AML patients co-acquire CSF3R and RUNX1 mutations, which play cooperative roles in the development of AML. To establish an in vitro model of leukemogenesis, we utilized bone marrow lin- cells from transgenic C57BL/6-d715 Csf3r mice expressing a CN patient-mimicking truncated CSF3R mutation. We transduced these cells with vectors encoding RUNX1 wild type (WT) or RUNX1 mutant proteins carrying the R139G or R174L mutations. Cells transduced with these RUNX1 mutants showed diminished in vitro myeloid differentiation and elevated replating capacity, compared with those expressing WT RUNX1. mRNA expression analysis showed that cells transduced with the RUNX1 mutants exhibited hyperactivation of inflammatory signaling and innate immunity pathways, including IL-6, TLR, NF-kappaB, IFN, and TREM1 signaling. These data suggest that the expression of mutated RUNX1 in a CSF3R-mutated background may activate the pro-inflammatory cell state and inhibit myeloid differentiation.

Molecular Landscape of Acute Myeloid Leukemia: Prognostic and Therapeutic Implications.

PURPOSE OF REVIEW: The field of acute myeloid leukemia (AML) has been revolutionized in recent years by the advent of high-throughput techniques, such as next-generation sequencing. In this review, we will discuss some of the recently identified mutations that have defined a new molecular landscape in this disease, as well as their prognostic, predictive, and therapeutic implications. RECENT FINDINGS: Recent studies have shown how many cases of AML evolve from a premalignant period of latency characterized by the accumulation of several mutations and the emergence of one or multiple dominant clones. The pattern of co-occurring mutations and cytogenetic abnormalities at diagnosis defines risk and can determine therapeutic approaches to induce remission. Besides the genetic landscape at diagnosis, the continued presence of particular gene mutations during or after treatment carries prognostic information that should further influence strategies to maintain remission in the long term. The recent progress made in AML research is a seminal example of how basic science can translate into improving clinical practice. Our ability to characterize the genomic landscape of individual patients has not only improved our ability to diagnose and prognosticate but is also bringing the promise of precision medicine to fruition in the field.

Pro-inflammatory cytokines favor the emergence of ETV6-RUNX1-positive pre-leukemic cells in a model of mesenchymal niche.

ETV6-RUNX1 (E/R) fusion gene, arising in utero from translocation t(12;21)(p13:q22), is the most frequent alteration in childhood acute lymphoblastic leukemia (ALL). However, E/R is insufficient to cause overt leukemia since it generates a clinically silent pre-leukemic clone which persists in the bone marrow but fails to out-compete normal progenitors. Conversely, pre-leukemic cells show increased susceptibility to transformation following additional genetic insults. Infections/inflammation are the most accredited triggers for mutations accumulation and leukemic transformation in E/R+ pre-leukemic cells. However, precisely how E/R and inflammation interact in promoting leukemia is still poorly understood. Here we demonstrate that IL6/TNFα/ILß pro-inflammatory cytokines cooperate with BM-MSC in promoting the emergence of E/R+ Ba/F3 over their normal counterparts by differentially affecting their proliferation and survival. Moreover, IL6/TNFα/ILß-stimulated BM-MSC strongly attract E/R+ Ba/F3 in a CXCR2-dependent manner. Interestingly, E/R-expressing human CD34+ IL7R+ progenitors, a putative population for leukemia initiation during development, were preserved in the presence of BM-MSC and IL6/TNFα/ILß compared to their normal counterparts. Finally, the extent of DNA damage increases within the inflamed niche in both control and E/R-expressing Ba/F3, potentially leading to transformation in the apoptosis-resistant pre-leukemic clone. Overall, our data provide new mechanistic insights into childhood ALL pathogenesis.

Insights into the prenatal origin of childhood acute lymphoblastic leukemia.

Pediatric acute lymphoblastic leukemia (ALL) is defined by recurrent chromosomal aberrations including hyperdiploidy and chromosomal translocations. Many of these aberrations originate in utero and the cells transform in early childhood through acquired secondary mutations. In this review, we will discuss the most common prenatal lesions that can lead to childhood ALL, with a special emphasis on the most common translocation in childhood ALL, t(12;21), which results in the ETV6-RUNX1 gene fusion. The ETV6-RUNX1 fusion arises prenatally and at a 500-fold higher frequency than the corresponding ALL. Even though the findings regarding the frequency of ETV6-RUNX1 were originally challenged, newer studies have confirmed the higher frequency. The prenatal origin has also been proven for other gene fusions, including KMT2A, the translocations t(1;19) and t(9;22) leading to TCF3-PBX1 and BCR-ABL1, respectively, as well as high hyperdiploidy. For most of these aberrations, there is evidence for more frequent occurrence than the corresponding leukemia incidences. We will briefly discuss what is known about the cells of origin, the mechanisms of leukemic transformation through lack of immunosurveillance, and why only a part of the carriers develops ALL.

Mechanisms of Progression of Myeloid Preleukemia to Transformed Myeloid Leukemia in Children with Down Syndrome.

Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS.

Clonal hematopoiesis and preleukemia-Genetics, biology, and clinical implications.

Myeloid neoplasms including myelodysplastic syndromes and acute myeloid leukemia (AML) originate from hematopoietic stem cells through sequential acquisition of genetic and epigenetic alterations that ultimately cause the disease-specific phenotype of impaired differentiation and increased proliferation. It has become clear that preleukemic clonal hematopoiesis (CH), characterized by an expansion of stem and progenitor cells that carry somatic mutations but are still capable of normal differentiation, can precede the development of clinically overt myeloid neoplasia by many years. CH commonly develops in the aging hematopoietic system, yet progression to myelodysplasia or AML is rare. The discovery that myeloid neoplasms frequently develop from premalignant precursor conditions that are detectable in many healthy individuals has important consequences for the diagnosis, and potentially for the treatment of these disorders. In this review, we summarize the current knowledge on CH as a precursor of myeloid cancers and the implications of CH-related gene mutations in the diagnostic workup of patients with suspected myelodysplastic syndrome. We will discuss the risk of progression associated with CH in healthy persons and in patients undergoing chemotherapy for a non-hematologic cancer, and the significance of CH in autologous and allogeneic stem cell transplantation. Finally, we will review the significance of preleukemic clones in AML and their persistence in patients who achieve a remission after chemotherapeutic treatment.

Ultra-Sensitive CSF3R Deep Sequencing in Patients With Severe Congenital Neutropenia.

High frequency of acquired CSF3R (colony stimulating factor 3 receptor, granulocyte) mutations has been described in patients with severe congenital neutropenia (CN) at pre-leukemia stage and overt acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Here, we report the establishment of an ultra-sensitive deep sequencing of a CSF3R segment encoding the intracellular "critical region" of the G-CSFR known to be mutated in CN-MDS/AML patients. Using this method, we achieved a mutant allele frequency (MAF) detection rate of 0.01%. We detected CSF3R mutations in CN patients with different genetic backgrounds, but not in patients with other types of bone marrow failure syndromes chronically treated with G-CSF (e.g., Shwachman-Diamond Syndrome). Comparison of CSF3R deep sequencing results of DNA and cDNA from the bone marrow and peripheral blood cells revealed the highest sensitivity of cDNA from the peripheral blood polymorphonuclear neutrophils. This approach enables the identification of low-frequency CSF3R mutant clones, increases sensitivity, and earlier detection of CSF3R mutations acquired during the course of leukemogenic evolution of pre-leukemia HSCs of CN patients. We suggest application of sequencing of the entire CSF3R gene at diagnosis to identify patients with inherited lost-of-function CSF3R mutations and annual ultra-deep sequencing of the critical region of CSF3R to monitor acquisition of CSF3R mutations.

Preleukemia and Leukemia-Initiating Cell Activity in inv(16) Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a collection of hematologic malignancies with specific driver mutations that direct the pathology of the disease. The understanding of the origin and function of these mutations at early stages of transformation is critical to understand the etiology of the disease and for the design of effective therapies. The chromosome inversion inv(16) is thought to arise as a founding mutation in a hematopoietic stem cell (HSC) to produce preleukemic HSCs (preL-HSCs) with myeloid bias and differentiation block, and predisposed to AML. Studies in mice and human AML cells have established that inv(16) AML follows a clonal evolution model, in which preL-HSCs expressing the fusion protein CBFß-SMMHC persist asymptomatic in the bone marrow. The emerging leukemia-initiating cells (LICs) are composed by the inv(16) and a heterogeneous set of mutations. In this review, we will discuss the current understanding of inv(16) preleukemia development, and the function of CBFß-SMMHC related to preleukemia progression and LIC activity. We also discuss important open mechanistic questions in the etiology of inv(16) AML.

IFN-γ directly inhibits murine B-cell precursor leukemia-initiating cell proliferation early in life.

The early-life immune environment has been implicated as a modulator of acute lymphoblastic leukemia (ALL) development in children, with infection being associated with significant changes in ALL risk. Furthermore, polymorphisms in several cytokine genes, including IL-10 and IFN-γ, are associated with leukemia development. However, the mechanisms and timing of these influences remain unknown. Here, we use the Eµ-ret transgenic mouse model of B-cell precursor ALL to assess the influence of IFN-γ on the early-life burden of leukemia-initiating cells. The absence of IFN-γ activity resulted in greater numbers of leukemia-initiating cells early in life and was associated with accelerated leukemia onset. The leukemia-initiating cells from IFN-γ-knockout mice had reduced suppressor of cytokine signaling (SOCS-1) expression, were significantly more sensitive to IFN-γ, and exhibited more rapid expansion in vivo than their wild-type counterparts. However, sensitivity to this inhibitory pathway was lost in fully transformed IFN-γ-knockout leukemia cells. These results demonstrate that the influence of IFN-γ on ALL progression may not be mediated by selection of nascent transformed cells but rather through a general SOCS-mediated reduction in B-cell precursor proliferation. Thus, while cytokine levels may influence leukemia at multiple points during disease progression, our study indicates a significant early influence of basal, infection-independent cytokine production on leukemogenesis.